Abstract

Short-term synaptic facilitation plays an important role in information processing in the central nervous system. Although the crucial requirement of presynaptic Ca2+ in the expression of this plasticity has been known for decades, the molecular mechanisms underlying the plasticity remain controversial. Here, we show that presynaptic metabotropic glutamate receptors (mGluRs) bind and release Munc18-1 (also known as rbSec1/nSec1), an essential protein for synaptic transmission, in a Ca2+-dependent manner, whose actions decrease and increase synaptic vesicle release, respectively. We found that mGluR4 bound Munc18-1 with an EC50 for Ca2+ of 168 nM, close to the resting Ca2+ concentration, and that the interaction was disrupted by Ca2+-activated calmodulin (CaM) at higher concentrations of Ca2+. Consistently, the Munc18-1-interacting domain of mGluR4 suppressed both dense-core vesicle secretion from permeabilized PC12 cells and synaptic transmission in neuronal cells. Furthermore, this domain was sufficient to induce paired-pulse facilitation. Obviously, the role of mGluR4 in these processes was independent of its classical function of activation by glutamate. On the basis of these experimental data, we propose the following model: When neurons are not active, Munc18-1 is sequestered by mGluR4, and therefore the basal synaptic transmission is kept low. After the action potential, the increase in the Ca2+ level activates CaM, which in turn liberates Munc18-1 from mGluR4, causing short-term synaptic facilitation. Our findings unite and provide a new insight into receptor signaling and vesicular transport, which are pivotal activities involved in a variety of cellular processes.

Short-term synaptic facilitation is a form of synaptic enhancement of presynaptic origin and is prominent within the hundreds of milliseconds time scale (1). It can be seen with pairs of stimuli, in which the second stimulus evokes more transmitter release than the first (1). Its expression is attributed to the effects of a residual elevation in presynaptic Ca2+ after previous neuronal activities (1). Although short-term facilitation is fundamental to information processing in the central nervous system, what links Ca2+ to this synaptic enhancement remains controversial (1, 2).

Presynaptic group III metabotropic glutamate receptors (mGluR4, mGluR7, and mGluR8) are expressed in the presynaptic active zone and reduce synaptic vesicle release on stimulation by agonists (3–6). A prominent enigma concerning the function of group III mGluRs is depicted clearly in the case of mGluR4: Short-term synaptic facilitation is impaired in mGluR4-KO mice, although the expression of the plasticity is not reduced by pharmacological blockade of mGluR4 (6, 7). These findings suggest the existence of some ligand-independent function of the receptor linked to synaptic vesicle release (6). Recently, we and others showed Ca2+-dependent calmodulin (CaM) binding to the intracellular C-terminal tail (ct) of group III mGluRs (8, 9). Because the affinity of CaM for Ca2+, ≈1 μM (10), corresponds to the required residual Ca2+ level for the expression of short-term facilitation, we assumed the existence of a molecule that can bind to the ct region of group III mGluRs and whose interaction with the receptor is regulated by Ca2+-activated CaM. Such a molecule, if it exists, would be a good candidate for mGluR-mediated expression of short-term synaptic facilitation (1). Herein, we show that presynaptic mGluRs sequester Munc18-1 (11) at the basal Ca2+ level and release it through interaction with Ca2+-activated CaM at the elevated Ca2+ range and that these actions are responsible for the respective basal neurotransmission and neuronal-activity-dependent increase in neurotransmission. Our findings reveal how Ca2+ acts in short-term facilitation through a ligand-independent signaling mechanism of mGluR.

Results and Discussion

We searched for proteins interacting with mGluR7 from a detergent-solubilized synaptosomal fraction of mouse brain in the presence of a calcium chelator, EGTA, to eliminate the binding of CaM to mGluR7. We found that GST-ct-mGluR7, but not GST alone, bound Munc18-1, which is a protein essential for neurotransmission (11) and which promotes SNARE-mediated vesicle fusion in a defined fusion system in which SNAREs are reconstituted in liposomes (ref. 12; Fig. 1A). Coimmunoprecipitation and in vitro binding experiments indicated that mGluR7 or mGluR4 directly interacted with Munc18-1 in the brain (Fig. 1B and C). Because Munc18-1 was retained more efficiently by GST-ct-mGluR4 (Fig. 1C), we focused on the interaction of Munc18-1 and mGluR4 in subsequent experiments.

Interaction of Munc18-1 with group III metabotropic glutamate receptors (mGluRs). (A) Affinity purification of Munc18-1 from a brain lysate by GST-ct-mGluR7. The arrow indicates the position of Munc18-1, as revealed by MALDI-TOF/MS. (B) Coimmunoprecipitation of Munc18-1 with mGluR7 (Left) or mGluR4 (Right) from a brain lysate: lysate, extract used for immunoprecipitation; IB, immunoblot; IP, immunoprecipitation. (C and D) Specific interaction of Munc18-1 with presynaptic group III mGluRs. In this and the following experiments, bound Munc18-1 or its mutants were detected by immunoblotting with anti-His6. (E) Domain 1 of Munc18-1 binds to ct-mGluR4: Full, full-length; D1, domain 1; D23, domain 2 plus 3 of Munc18-1. (F) Domain 1 of Munc18-1 (M18D1) reduces the ct-mGluR4/Munc18-1 binding. (G) Syntaxin inhibits the ct-mGluR4/Munc18-1 binding. (H) Munc18-1 binds to the membrane-proximal region of ct-mGluR4: ct-mGluR4–1, amino acids 848–889; ct-mGluR4-2, amino acids 890–912 of mGluR4. (I) Calmodulin (CaM) inhibits ct-mGluR4/Munc18-1 binding at a Ca2+ concentration of 1 μM. Calmodulin was detected with anti-CaM antibody (anti-CaM). In C–I, Ponceau S- or Coomassie-stained gels below the immunoblots show comparable amounts of the GST or GST fusion proteins immobilized on glutathione Sepharose 4B beads. Lanes labeled “C” contained 0.1 μg of proteins run as controls for blotting.

Group II mGluRs (mGluR2 and mGluR3) also are expressed presynaptically and modulate synaptic transmission (3–5). In contrast to group III mGluRs, these group II ones are not localized in the active zone but in preterminal portions of axons (5) and do not bind CaM (8). Munc18-1 did not bind to this group of mGluRs (Fig. 1D). This finding indicates the specificity of the interaction and correlates well with the site of action of both group III mGluRs and Munc18-1 at the active zone.

Munc18-1 was originally isolated as a binding partner of syntaxin 1, a core constituent of the SNARE complex crucial for synaptic vesicle exocytosis (11). We wondered whether mGluR4 is tethered to the SNARE through Munc18-1 or whether it functions separately. The structure of Munc18-1 reveals that it can be divided into three domains (13). Domain 1 of Munc18-1 (M18D1) is responsible for the binding to syntaxin 1 (13–15); M18D1 was sufficient for the interaction with ct-mGluR4 (Fig. 1E) and hindered the binding between ct-mGluR4 and Munc18-1 (Fig. 1F). These results suggest that Munc18-1 interacts with mGluR4 and syntaxin 1 in a similar fashion. Accordingly, the presence of syntaxin 1 disrupted the binding between Munc18-1 and mGluR4 (Fig. 1G).

Calmodulin binds to the membrane-proximal region of group III ct-mGluRs (8, 9). Munc18-1 bound to this region of ct-mGluR4 (ct-mGluR4–1; Fig. 1H), and the interaction was disrupted by Ca2+-activated CaM at a free Ca2+ concentration corresponding to that of residual Ca2+ (1) (Fig. 1I). Therefore, mGluR4 seems to compete with syntaxin 1 for Munc18-1 and regulates synaptic transmission by sensing Ca2+ via CaM.

Given the possibility that mGluR4, Munc18-1, and CaM constitute a sensing mechanism for Ca2+, we evaluated its operating range to determine its site of action in synaptic vesicle exocytosis. Although Munc18-1 does not have an apparent Ca2+-binding motif, the interaction between ct-mGluR4 and Munc18-1 was enhanced greatly in the presence of increasing concentrations of Ca2+, with saturation at 3 μM, but the interaction decreased at 1 mM (Fig. 2A). To our knowledge, this is the first demonstration of a Ca2+-dependent function of Sec1/Munc18 proteins (11), essential for vesicle trafficking in many routes employing SNARE complex formation. The EC50 of Ca2+ for the rising phase of the binding reaction was 168 nM (Fig. 2B), far below that for synaptotagmin I–syntaxin binding [>200 μM (16)] and the affinity of CaM for Ca2+ [≈1 μM (10)]. Consistent with this difference in the affinity for Ca2+, in the presence of CaM, more Munc18-1 was retained by ct-mGluR4 at a Ca2+ concentration of 0.1 μM than at 1 or 10 μM (Fig. 2C). Hence, the effective range of this Ca2+-sensing mechanism is <1 μM Ca2+ and contrasts well with that of synaptotagmin I, the Ca2+ sensor for rapid vesicle release (1, 11, 16). In the presence of CaM, mGluR4 seems to bind Munc18-1 at the resting Ca2+ level of 0.1 μM and release it above the Ca2+ level of 1 μM corresponding to the residual Ca2+ level after an action potential.

Ca2+-dependent binding and release of Munc18-1 by metabotropic glutamate receptor 4 (mGluR4). (A) Interaction of GST-ct-mGluR4 and Munc18-1 at increasing concentrations of Ca2+. Bound Munc18-1 was detected by immunoblotting with anti-His6. The Ponceau S-stained gel below the immunoblot shows comparable amounts of GST or GST-ct-mGluR4 immobilized on glutathione Sepharose 4B beads. The lanes labeled “C” contained 0.1 μg of Munc18-1 run as a control for blotting. Munc18-1 was not visible at 0 mM Ca2+ with the short-time exposure used to show Ca2+ dependency in Munc18-1 binding. However, as shown in C, a considerable amount of Munc18-1 was bound to GST-ct-mGluR4 even at 0 mM Ca2+, which is also apparent in this experiment with longer exposure. (B) Quantification of GST-ct-mGluR4/Munc18-1 interaction at increasing concentrations of Ca2+. Bound Munc18-1 at 3 μM Ca2+ was set to 100%, and that at 0 μM Ca2+ was set to 0%. Fitting the data with the Hill equation yielded an EC50 of 0.168 μM (95% C.I., 0.134 to 0.209). (C) In the presence of calmodulin (CaM), significantly more Munc18-1 was retained by ct-mGluR4 at a Ca2+ concentration of 0.1 μM (P < 0.01, Bonferroni post hoc test after one-way ANOVA). In both B and C, the results shown are the means ± SD (n = 2).

To evaluate the physiological significance of this Ca2+-sensing mechanism, the following issues are important to address: the effect of mGluR4/Munc18-1 binding on vesicle secretion, formation of the functional complex under physiological conditions, and its effect on synaptic transmission. In the following experiments, the first two issues were evaluated in a model system amenable to quantitative analysis, and the last one was examined by using natural neuronal cells devoid of mGluR4 activity.

To analyze the effect of mGluR4/Munc18-1 binding on Ca2+-triggered vesicle secretion, we used a model system using permeabilized PC12 cells (17). In this system, the same basic secretory machinery that is present in neurons, including the SNARE complex and Munc18-1, is conserved (17–19). Furthermore, the Ca2+ concentration can be controlled experimentally independently of Ca2+ channel activation, and exogenous proteins can be introduced quantitatively. In this assay, Ca2+-triggered exocytosis is independent of GTP-dependent exocytosis (17), and so we did not include GTP to exclude any GTP-dependent exocytosis. The GST-ct-mGluR4 (20 μM) suppressed exocytosis over a broad physiological range of Ca2+ concentrations (Fig. 3A), whereas GST-ct-mGluR2 was not effective (Fig. 3B). The GST-ct-mGluR4 inhibited the secretion dose-dependently, with an EC50 of 1 μM and a maximal inhibition of 40% (Fig. 3C). This potency of GST-ct-mGluR4 was close to that of the synaptotagmin VII C2A domain (20) and greater than the requirement of the C-terminal coil of SNAP-25 (21). Although CaM retained in this system has a limited stimulatory effect on the secretion, it has little or no effect on the secretion <0.3 or >3 μM Ca2+ (19), where GST-ct-mGluR4 induced prominent inhibition (Fig. 3A and C). Thus, the majority of the inhibition by GST-ct-mGluR4 reflected the effect of mGluR4/Munc18-1 binding, not mGluR4/CaM binding. These results indicate that mGluR4/Munc18-1 binding inhibits vesicle secretion independently of ion channel modulation and G protein cascades.

Inhibition of vesicle secretion by ct-mGluR4. (A and B) The Ca2+-triggered vesicle secretion from cracked PC12 cells in the presence of 20 μM each of GST-ct-mGluR4 (closed circle in A), GST-ct-mGluR2 (closed circle in B), or GST (open circle). The maximal Ca2+-dependent release of [3H]norepinephrine under control conditions run in parallel was set to 100%. (C) Dose-dependent suppression of Ca2+-triggered vesicle secretion by GST-ct-mGluR4. The Ca2+-dependent release of [3H]norepinephrine in the absence of GST fusion proteins was set to 100%: GST, open squares; GST-ct-mGluR2, open circles; GST-ct-mGluR4, closed circles. The free Ca2+ concentration was 3 μM, and the EC50 of GST-ct-mGluR4 was 1.05 μM (95% C.I., 0.569 to 1.94). Results shown are the means ± SD (n = 3).

We then studied the effects of the mGluR4/Munc18-1 interaction on synaptic transmission at cholinergic synapses formed between superior cervical ganglion neurons (SCGNs) in culture (22, 23). In these cells, mGluR4 is not expressed (24). We injected Munc18-1-interacting mGluR4 C-terminal peptide (mGluR4C, residues 849–889 corresponding to ct-mGluR4–1) instead of GST-ct-mGluR4 to permit its rapid diffusion to presynaptic terminals. With this approach, we could isolate the acute and purely intracellular function of the ct-mGluR4/Munc18-1 interaction without interference from the constitutive activity of mGluR4 (25) and compensatory changes caused by manipulation of gene expression levels. The injection of mGluR4C caused a gradual decrease in the amplitude of evoked excitatory postsynaptic potentials (EPSPs) (Fig. 4A–C). Thirty minutes after the start of injection, the mean EPSP amplitude decreased by 24 ± 4.1% (mGluR4C, n = 5; Fig. 4C). The time course and extent of inhibition were similar to those caused by the injection of M18D1 [21 ± 2.0% (M18D1, n = 5)] used to verify and monitor inhibition of Munc18-1-mediated secretion in these synapses (Fig. 4B and C). In control injections, scrambled mGluR4C peptide (SCB) caused no significant change in EPSP amplitudes (+1.9 ± 3.6%). These results are consistent with the idea that the mGluR4/Munc18-1 interaction is inhibitory to synaptic transmission.

Generally, a decrease in the initial probability of transmitter release leads to larger synaptic enhancement or reduction of synaptic depression (1, 2). From the above inhibitory role of mGluR4C in synaptic transmission and the operating range of the Ca2+-sensing mechanism (Fig. 2C), we assumed that this domain is responsible for the expression of short-term synaptic facilitation or at least reduces short-term synaptic depression. To verify this hypothesis, we examined the effect of mGluR4C on the expression of short-term synaptic facilitation by using a paired-pulse protocol (Fig. 4D and E). Depending on the interstimulus interval, SCGN synapses exhibited paired-pulse depression (PPD, ≤60 ms) or weak paired-pulse facilitation (PPF, 80–120 ms). The injection of mGluR4C suppressed the PPD, significantly at 30 ms of the paired-pulse interval (Fig. 4D; n = 7, three to five measurements for each) and induced PPF in some synapses (Fig. 4E; 5 of 26 measurements). Because mGluR4C had no effect on the expression of PPF (100 ms), mGluR4C seemed to introduce a facilitating property to SCGN synapses, which was apparent when original SCGN synapses exhibited PPD. These results are consistent with the idea that the absence of the ct region of mGluR4 accounts for the impaired expression of PPF in mGluR4-KO mice (7).

In the above experiments, we could not show quantitatively that the majority of the ct-mGluR4-mediated effects on synaptic transmission are caused by the sequestration of Munc18-1 and not of CaM, which has pleiotropic effects on cellular functions. However, under physiological conditions, the assumption that because more CaM is present in the nerve terminals than mGluR4 there is not enough capacity for mGluR4 to cause significant alteration in the free concentration of CaM would be rational. Accordingly, a significant fraction of the observed effects of ct-mGluR4 on synaptic transmission would be described in the following model: When neurons are not active, Munc18-1 is sequestered by mGluR4, and therefore, the basal synaptic transmission is kept low. After the action potential, the increase in the Ca2+ level activates CaM, which in turn liberates Munc18-1 from mGluR4, causing short-term synaptic facilitation. This Ca2+-sensing mechanism reveals a function of mGluR4 in the expression of short-term facilitation and explains the discrepancy between pharmacological blockade and gene targeting of mGluR4, revealed clearly at the parallel fiber synapses onto cerebellar Purkinje cells (6, 7). Another interesting implication of our findings is that the presence of mGluR4 is responsible for the expression of certain synaptic properties [e.g., mGluR4-positive facilitating parallel fiber synapses and mGluR4-negative depressing climbing fiber synapses onto the same cerebellar Purkinje cells (1, 2, 26)]. Although Munc18-1 is known to be an essential protein for synaptic transmission, evidence for both positive and negative roles of Munc18-1 in synaptic transmission has been reported (11). If Munc18-1 has a negative role in synaptic transmission, then our data would mean that the ct-mGluR4/Munc18-1 interaction augments the inhibitory function of Munc18-1. In this case, disruption of this negative complex by Ca2+-activated CaM would relieve Munc18-1-mediated inhibition and consequently lead to synaptic facilitation. Thus, no matter whether Munc18-1 has a positive or negative role in synaptic vesicle release, the Ca2+-sensing system presented here would operate in the expression of short-term synaptic facilitation. Further characterization of interacting partners of presynaptic group III mGluRs or other receptors and studies aimed at manipulation of the interaction would not only elucidate the mechanisms of diversity in synaptic properties but also provide a basis for development of new therapeutic approaches to neuronal disorders.

Materials and Methods

The animals were treated as approved by the Animal Research Committee of Kyoto University for the ethical use of experimental animals.

Antibodies.

Polyclonal antibodies against mGluR4 and mGluR7 were prepared as described in ref. 5. Normal rabbit IgG was purified from rabbit serum (Invitrogen) by using Protein A Sepharose (GE Healthcare). Anti-His6 was from Qiagen, and anti-CaM was from Millipore. Anti-Munc18-1 was from Becton Dickinson.

Plasmid Vectors and Protein Purification.

Constructs expressing the ct-mGluRs were made in the pGEX4T-1 vector. Munc18-1 was amplified by PCR using Mouse Brain Marathon-Ready cDNA (Clontech) as a template. Truncations of Munc18-1 were made by PCR. The pQE80L vector (Qiagen) was used for the expression of Munc18-1 and its mutants. The full cytoplasmic domain of syntaxin 1a (amino acids 1–266) was amplified from rat brain total RNA by RT-PCR. This fragment and a nucleotide sequence for an N-terminal HA tag were cloned together into a pGEX6P-1 vector for bacterial expression. Integrity of the PCR products was confirmed by DNA sequencing of both strands of all constructs. The amino acid residues of ct-mGluRs and database accession numbers are as follows: ct-mGluR2, Gln 820–Leu 872 (NP_001099181.1); ct-mGluR3, Gln 829–Leu 879 (NP_001099182.1); ct-mGluR4, His 848–Ile 912 (NP_073157.1); ct-mGluR4-1, His 848–Leu 889; ct-mGluR4-2, Cys 890–Ile 912; ct-mGluR7, His 851–Ile 915 (NP_112302.1). Munc18-1 domain 1 and domain 2 plus 3 are Met 1–Asn 134 and Ile 135–Ser 594 of NP_033321.2, respectively. The full cytoplasmic domain of syntaxin 1a corresponds to Met 1–Ile 266 of NP_446240. The amplified coding sequence of Munc18-1 was the same as that of AF326563 in GenBank, whereas the codon for Ala 86 of syntaxin 1a was GCA instead of GCG in GenBank (NM_053788).

The GST fusion proteins and His-tagged proteins were expressed in Escherichia coli strain BL21 and purified by use of glutathione Sepharose 4B beads (GE Healthcare) and TALON Metal Affinity Resin (Clontech), respectively. The full cytoplasmic domain of syntaxin 1a was liberated from GST by PreScision Protease (GE Healthcare) according to the instructions from the manufacturer. Protein concentration was determined by DC Protein Assay Kit (Bio-Rad) using BSA as a standard.

Affinity Purification of ct-mGluR7-Interacting Proteins.

Synaptosomes from whole mouse brains were prepared as described in ref. 27. The synaptosomes (≈20 mg) were first solubilized for 1 h at 4 °C in 1% SDS and then diluted with five volumes of cold Triton X-100 (TX) buffer [20 mM Hepes–NaOH, pH 7.4, containing 2% Triton X-100, 150 mM NaCl, 5 mM EGTA, and complete protease inhibitor cocktail (Roche)]. After centrifugation at 100,000 × g for 1 h at 4 °C, the supernatant was passed through a 1-mL column of glutathione Sepharose 4B to reduce nonspecific binding. One milliliter of the cleared supernatant was incubated for 4 h at 4 °C with 50 μL of glutathione Sepharose 4B beads previously coated with 100 μg of GST or GST-ct-mGluR7. After having been washed four times with a 1:5 mixture of 1% SDS and TX buffer, the bound proteins were separated on an SDS/PAGE gel and stained with Coomassie Blue R-250. We searched for specifically interacting proteins by using MALDI-TOF/MS (28) to compare proteins in each band with those in the corresponding area of the control lane.

Immunoprecipitation.

Synaptosomes from mouse brains were solubilized for 1 h at 4 °C in immunoprecipitation buffer (20 mM Hepes–NaOH, pH 7.4, containing 1% CHAPS, 150 mM NaCl, and complete protease inhibitor cocktail). After centrifugation at 100,000 × g for 1 h, the supernatant (3.2 mg of protein) was incubated with 5 μg of anti-mGluR7 IgG or normal rabbit IgG for 2 h at 4 °C. Immunocomplexes were recovered by incubation with 20 μL of Protein A Sepharose for 2 h at 4 °C. After elution with SDS/PAGE loading buffer and separation by SDS/PAGE, bound Munc18-1 was detected by immunoblotting with anti-Munc18-1 antibody. Immunoprecipitation using anti-mGluR4 IgG was conducted similarly. In this case, synaptosomes from rat cerebella were used, and the concentration of Hepes–NaOH in the immunoprecipitation buffer was 25 mM.

In Vitro Binding Assay.

The GST or GST fusion proteins (10 μg of each) were immobilized on glutathione Sepharose 4B beads (20 μL). The N-terminally His-tagged Munc18-1 or its mutants (15 μg) were used for in vitro binding assays as described in ref. 8. For CaM competition experiments, 2 mM EGTA was added to the buffer, and the free Ca2+ concentration was adjusted by using the WEBMAXC program (29). Five micrograms of Munc18-1 and a 10-fold molar excess of CaM were used in the competition experiments. Quantification of bound Munc18-1 was conducted by using a LAS-1000 luminescent image analyzer (Fuji) with known amounts of Munc18-1 as the standard. The EC50 was calculated by fitting the data points to a sigmoidal dose–response equation by using Prism software (GraphPad).

Cracked PC12 Cell Secretion assays.

Cracked PC12 cell secretion assays were done with freeze–thaw permeabilized PC12 cells (17). The complete reaction mixture in 1.5-mL microcentrifuge tubes (100 μL final volume) contained cell ghosts, 2 mM ATP, 2 mM MgCl2, 10 μL of rat brain cytosol (10 mg/mL) in KGlu buffer (20 mM Hepes–NaOH, pH 7.2, containing 120 mM potassium glutamate, 20 mM potassium acetate, and 2 mM EGTA), and various concentrations of CaCl2 and recombinant proteins. Proteins used in cracked cell assays were dialyzed extensively against appropriate buffers to make the final composition of the reaction mixtures the same as that of the KGlu buffer. Free calcium concentrations were calculated by using WEBMAXC. Reactions were incubated for 30 min at 30 °C. The EC50 was calculated by fitting the data points to a sigmoidal dose–response equation by using Prism software.

Synaptic Transmission between SCGNs.

Culturing of SCGNs, EPSP recording, and injection of recombinant proteins and synthetic peptides were performed as described in refs. 22 and 23. The concentration of peptide in the pipette was 160 μM (M18D1) or 1 mM (mGluR4C and SCB). Collected electrophysiological data using software written by the late L. Tauc (Centre National de la Recherche Scientifique, Gif-sur-Yvette, France) were analyzed with Origin 8 (Microcal Software). The EPSPs were recorded every 10 s, and the peak amplitudes of EPSPs were averaged. The resultant values were smoothed by an eight-point moving average algorithm and plotted against recording time, with t = 0 indicating the start of the presynaptic injection of 3 min in duration. For Fig. 4C, data at 30 min after injection from each experiment were averaged, and statistical significance was determined by Bonferroni post hoc test after one-way ANOVA. Short-term synaptic plasticities were examined at 17 min postinjection. For comparison of paired-pulse response ratios, EPSP amplitudes from three to five recordings were averaged, and the resultant values from seven pairs were averaged.

Acknowledgments

We thank A. Kakizuka and S. Hori for critical discussions and reading of the manuscript. This work was funded by research grants from the Ministry of Education, Science, and Culture of Japan.

Footnotes

1To whom correspondence may be addressed. E-mail: yoshiaki{at}phy.med.kyoto-u.ac.jp or snakanis{at}obi.or.jp

Author contributions: Y.N. and S.N. designed research; Y.N., S.M., and K.O. performed research; Y.N. contributed new reagents/analytic tools; Y.N. and S.M. analyzed data; and Y.N. and S.N. wrote the paper.

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